A successful vaccine against HIV will likely require a strong, broad memory T cell and neutralizing humoral immune response. It is not clear how to stimulate such a broad immune response and strategies to date have not been successful. Thus, it is seems reasonable to pursue novel vaccine strategies in the well characterized feline immunodeficiency virus (FIV)/cat model. Listeria monocytogenes (LM) is a Gram positive, facultative intracellular bacterium that can be used to deliver foreign antigens to antigen presenting cells resulting in induction of a strong cell-mediated immune response. We have generated recombinant LM that express FIV Gag from sequence inserted into the bacterial genome and delivers a DNA vaccine for eukaryotic expression of Env proteins. We have shown a single oral immunization with this vaccine vector induced a strong cell-mediated response and high titer vaginal IgA. Upon vaginal challenge with pathogenic FIV, significant protection was demonstrated. Here we propose to further develop and test a novel prime-boost immunization strategy that would combine the benefits of two novel vaccine strategies. In specific aim 1, we will generate Gag and Pol expressing recombinant LM using two different attenuated Listeria isolates. After in vitro validation, we will test these recombinant LM for safety and immunogenicity in vivo. The optimal dose for oral immunization will also be determined. In the second specific aim, we will develop and test a novel DNA vaccine that will be used as the prime. The prime will consist of a PLG-microsphere encapsulated DNA vaccine containing FIV gag, pol, and env sequences. In addition, synthetic adjuvants will be added to the microspheres to specifically stimulate toll-like receptor (TLR) 4 or TLR 9. The prime will be given either orally or parenterally to compare the two routes. Immunogenicity will be proven in vivo. In the third specific aim, the optimized prime and optimized boost will be tested in a three-arm, challenge study. This strategy will determine if a microencapsulated DNA vaccine with a TLR-specific adjuvant, can effectively prime the immune system for a subsequent oral boost with recombinant LM resulting in a protective immune response at mucosal surfaces. These studies will be performed in the FIV/cat model that allows challenge by vaginal exposure to cell-associated and cell-free virus.